Treatment with dopamine (DA) D2 receptor antagonist significantly accelerated mobilization of exogenously transplanted MSCs towards wound bed.
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(A) Flow cytometric analysis of DA D2 receptors in murine MSCs. To confirm that CD34− CD45− CD105+ SSEA-4+ cells express DA D2 receptors, in vitro expanded Linneg bone marrow cell population (containing CD45−, CD11b− cell populations) were initially gated to exclude dead cells and debris and then the CD34− cell population were selected and these CD34− CD45− cells were evaluated for presence of CD105, SSEA-4 and DA D2 receptors. Results showed that almost 86% cells of the total MSC population (both CD34− CD45− CD105+ cells and CD34− CD45− SSEA-4+ cells) express DA D2 receptors on their surfaces. (B) Western blot analysis of DA D2 receptors in murine BM-MSCs. H4 neuroglioma cell line and sarcoma-180 (S-180) tumor cells were used as positive and negative controls, respectively. (C) Effect of DA D2 receptor antagonist treatment on mobilization of exogenously transplanted MSCs towards wound site. MSCs were labeled with BrdU in culture and injected into the tail vein of both control and eticlopride treated back skin-injured mice. After completion of eticlopride treatment, at 6th day the skin from the wound area was collected and analyzed by immunohistochemistry using BrdU labeling and detection kit (Roche Applied Science). Significantly higher number of BrdU positive transplanted MSCs are located in the wound bed of eticlopride treated group than vehicle treated control group, showing DA D2 receptor antagonist treatment has significant positive effect on mobilization of exogenously transplanted MSCs towards wound site. Original magnifications, ×200. Results are representative of six separate experiments each yielding similar results. (D) Graphical representation showing significantly higher number of exogenously transplanted MSCs (BrdU positive cells) in wound bed of eticlopride treated groups compared to vehicle treated controls at day 6 post wounding (*, P<0.05). Number of transplanted MSCs cells was measured by counting the number of BrdU positive cells in 10 randomly chosen high power microscopic fields within the sections.